The present invention is directed to quantitative testing apparatus and methods which may be used for a wide range of diagnostic and prognostic evaluations of various cells, antigens, or other biological materials taken from the human body. However, for purposes of illustration and ease of understanding, the invention will be disclosed in conjunction with its preferred use, which is the quantitative measurement of cellular DNA for the purpose of cancer diagnosis and prognosis. More specifically, the present invention is directed to methods and apparatus for interactive image analysis which are adapted to analyze and quantify the DNA in different classes of specimen cells taken from a human body.
The current state of the art in the pathology laboratory for measuring the DNA content of a cell is by visual observation. A pathologist observes through a microscope primarily the shape and texture of suspected cancer cells and then classifies the cells into a normal category or into one of several abnormal or cancer categories. Such evaluations are very subjective and can not differentiate and precisely quantify small changes in DNA within individual cells or in very small populations of abnormal cells. These small changes may represent an incipient stage of cancer or a change in cell structure due to treatment of the cancer by chemotherapy or radiation. Such small changes are, therefore, important in the diagnosis and prognosis of these diseases.
However, the advantage in diagnosis and/or prognosis of abnormal ploidy distributions that a pathologist viewing a specimen under a microscope has is the discerning expertise of a skilled person in classifying cells as normal or abnormal. There is an innate human ability to make relatively quick infinite gradations of classification, i.e., almost normal, slightly abnormal, etc. On the other hand, the classification and measurement of cell features and parameters manually by a pathologist on a cell-by-cell basis is extremely tedious and time consuming. Statistical analysis of such cell data taken by hand is relatively difficult because each record has to be entered and then processed. For different records, taken at different times, and under varying conditions broad statistical categorizations may be unreliable.
The alternative is automated cell analysis where the pathologist uses specialized equipment to perform the analysis. In automatic cell analysis, such as that accomplished by a flow cytometer, mass tests are performed in gross on a specimen cell population without a researcher being able to exclude or include certain data of the population. The specimen is measured "as is" without really knowing what cells are being measured and how many. Important single cell data or data from relatively small groups of cells are lost in the overall averaging of a specimen. Further, relatively large amounts of a specimen have to be used to provide results from these tests and the sample is consumed.
Although there are commercially available general purpose flow cytometers, they are very expensive and can handle only liquid blood specimens or tissue disaggregations. These cytometers are incapable of working on standard tissue sections or using conventional microscope slides which are the preferred specimen forms of pathology laboratories. Additionally, a flow cytometer does not allow for the analysis of morphological features of cells such as texture, size and shape of cell nuclei and alterations in the nuclear-to-cytoplasmic ratios of cells.
The methods and apparatus illustrated in the referenced Bacus applications have solved these and other problems relating to the analysis of various features and parameters of cell objects. Bacus discloses a measurement method and apparatus which can acquire accurate quantitative data concerning a plurality of individual cells very quickly by an interactive process with a pathologist or an operator.
The Bacus apparatus provides means for displaying on a video monitor an image of a group of cells from a field of a microscope slide. The image is further digitized and stored in a memory of the apparatus. From the digitized image, a processor means identifies each possible cell object automatically by a pattern recognition technique. An interactive program allows the operator to point to each object or cell in succession and make morphological decisions for classification and measurements concerning each. For quantitative DNA analysis, the measurement is of the optical density of the cell object and the classification is by a pathologist as to whether the cell appears normal or cancerous. The decisions include whether to accept or reject a particular cell for further measurement and processing. The cell object, if selected, can then also be classified into one of several classifications for later statistical analysis. The apparatus further has means which permit the classification and storing of more than one image.
When the apparatus is used for DNA analysis, tissue and cell specimens are applied to a slide which is then stained with a specific stain that combines proportionately with the DNA and essentially renders invisible the remainder of the cell so that the image analysis apparatus can measure the optical density of the DNA which is concentrated in the nucleus of the cell. The stain associates with the DNA to provide a detailed nuclear structure and pattern which may be visually observed and interpreted by the pathologist using the apparatus for classification. The amount of DNA in the malignant cells is substantially greater than that for normal cells because the malignant cells are usually dividing and replicating rapidly or the malignant cells have abnormal numbers of chromosomes or have defective chromosomes.
The Bacus apparatus can not only detect minute alterations in the nucleus by providing a real and accurate measurement of the DNA mass in picograms but also can measure and quantify the amount of DNA and relate it to stored statistical analyses to aid in diagnosis. More specifically, the invention allows an iterative analysis of specimen population cells and provides a histogram or other statistical display of the population distribution of the cells with respect to their DNA content and with respect to a standard DNA for normal cells so that subtle shifts in population distribution can be readily understood. To this end cell nuclei images are not only acquired and stored but the data therefrom can be integrated with other statistical data to provide multivariable analysis, discrimination of cells, histograms, and scattergrams of cells or cell populations.
While the methods and apparatus described above are extremely advantageous and advance the art of aneuploidy analysis by image processing, they are not as sensitive as they could be. With the progress in measuring the quantity and distribution of DNA in a cellular population, there has come the need to further refine and sensitize that analysis and characterization process. One area in which sensitivity of the above described process can be improved is in the operator classification of cell types.
The previous apparatus of Bacus relies mainly on the pathologist or operator to make a subjective judgement concerning the classification of cell types, and whether they are to be classified at all. This is a principal advantage of the apparatus where the expertise of the pathologist in discerning cell types is automated and measurement of specified parameters of the chosen cells is accurately made. However, it has been learned that different cell types which are really quite different structurally appear morphologically similar under the microscope.
This is particularly true when the nuclear DNA has been enhanced by Feulgen staining. Such nuclear staining is for the purpose of enhancing the optical characteristics of the nuclei of the cells which contain the DNA, but that necessarily de-emphasizes the visual characteristics of the cytoplasm in the rest of the cell outside of the nucleus. The result is to allow easier image analysis and precise measurement of the DNA of the nuclear material, but at the same time this enhancement causes the loss of the visual morphological characteristics of the cytoplasm which a pathologist might use to distinguish one type of cell from another. Additionally, there are different cell types, which it is advantageous to classify separately, but which provide no or only faint visual clues as to their differences.
Thus, there is the need to alert a pathologist classifying the cell populations for DNA analysis with the Bacus instrument about the different cell types, whether by optical enhancement or otherwise. A more definitive mechanism would be the use of some demonstrable marker on the cells themselves by which the pathologist can objectively separate the various cell types. There are known in the art many optical enhancement or marking techniques for cell populations, including those described in the above referenced Bacus applications. For example, since the advent of monoclonal antibody production, numerous antibodies have been developed which are specific for cellular components, either in the cytoplasm, nucleus or on the cell membrane. Some have already been used to type cells in pathology to assist in the definition of the cell of origin of a number of tumors where subjective morphology is equivocal.
Among the most notable of these antibodies are antibodies to Leukocyte Common Antigens, which identify inflammatory cells, and antibodies to a family of cytoplasmic structural proteins called cytokeratins which identify cells arising from epithelial structures. Other antibodies to cytoplasmic components such as intermediate filaments can be utilized to identify cells which provide structural support, the so called stromal cells. In addition, numerous antibodies exist which are more specifically related to individual tumor types.
However, further optical enhancement of the cytoplasm for different types of cells is problematic in view of the current DNA staining technique. There are many difficulties, the least of which is that an optical enhancement factor for the cytoplasm should be compatible with the present imaging techniques using computer analysis of optical density and be required to provide such compatibility without impairing the sensitivity of the imaging techniques for the present nuclear staining. Chemical compatibility with the present Feulgen staining technique also presents a major hurdle. Optical enhancement of the cytoplasm after Feulgen staining of the DNA is substantially unavailable because the Feulgen process is destructive of the cell cytoplasm and changes the way it appears normally. However, prior optical enhancement of the cytoplasm is equally as difficult because the Feulgen staining process is caustic with the use of highly acidic reagents which can easily destroy other optical enhancement factors. Moreover, if done prior to Feulgen staining, the optical enhancement process of the cytoplasm cannot affect the nuclear material in a manner such that the result of the subsequent Feulgen staining will be changed.